The broad and long term objective of the research described in this application is the elucidation of the structural, electronic, and chemical properties of metal-containing coordination sites of copper enzymes that reversibly bind and/or activate dioxygen or dioxygen analogs (nitric oxide). The methodology is that of the synthetic analog approach to the active sites of metallobiomolecules, whereby low molecular weight complexes are synthesized and examined at a small molecule level of detail. The properties revealed are thus intrinsic to the metal complex uncoupled from the influences of the protein matrix. The proposed research is ultimately directed towards the assembly and characterization of trinuclear copper clusters that mimic the structure and reactivity of the trinuclear copper clusters found in blue oxidases, which are responsible for reduction of dioxygen to water. The mechanism of activation is dramatically different from that of the dinuclear copper centers of tyrosinases or hemocyanins, though a structural similarity exists. A full understanding of the dioxygen reactivity of synthetic, trimeric copper complexes will help clarify some of the existing structural disparities between crystallography and spectroscopy. This analysis will require study of, not only the trinuclear centers, but also the related monomeric and dimeric copper complexes. Ligand design and synthesis is an integral part of this research; development of synthetic ligand systems which have the requisite flexibility to accommodate the different geometric preferences of Cu-II and Cu-I, while still maintaining a predisposed organization, is a necessary goal of this proposal. The specific aims of the proposed research are as follows: (i) characterization of mu-eta2:eta2Cu2-O2-L2 copper complexes with simple monomeric ligands at low temperatures to define the minimum geometric and electronic constraints necessary for dioxygen binding and activation; (ii) stabilization of a synthetic, tetrahedral Cu-II species in a facial capping, trinitrogen ligand to provide well defined spectroscopic examples that can be used as spectroscopic benchmarks; (iii) characterization and stabilization of the reaction intermediates of nitric oxide and nitrite with tetrahedral Cu-II and Cu-I complexes with the intention of generating species relevant to the catalytic cycle of nitrite reductase; (iv) development of a modular synthetic approach to ligand assembly for the synthesis of trinuclear copper clusters designed to stabilize copper/dioxygen intermediates; (v) assembly of synthetic trinuclear copper clusters structurally similar to that observed in ascorbate oxidase with trinuclear ligands and characterization of their reactivity with dioxygen.